Evaluation of Intestinal Gene Expression

Total RNA was extracted from the jejunal mucosa sample with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in terms of the manufacturer’s instructions. The cDNA was synthesized with the TaKaRa reverse transcription kit (TaKaRa Biotechnology, Tokyo, Japan) following RNA quality and concentration measurement. The mRNA expression levels of Nrf2, HO-1, NF-κB, ZO-1, occludin, and claudin-1 in jejunal mucosa tissues were assessed using an ABI 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR green real-time PCR regent (TaKaRa Biotechnology, Tokyo, Japan) as previously described (26 (link)). Primer sequences are shown in Table S3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene for gene normalization and quantification. The relative mRNA abundances of the target genes were calculated using the threshold cycle (2^-ΔΔCt) method.